The members of the ubiquitously expressed E-protein subfamily of basic Helix-Loop-Helix (bHLH) transcription factors, E12/E47, SEF2-1 and HEB, have important roles as regulators of gene expression in various differentiation processes, including lymphocyte development and myogenesis. In myogenesis, E-proteins are proposed to function as obligate heterodimer partners for members of the MyoD family of muscle-specific bHLH transcription factors. The calcium ion (Ca2+) is a universal cellular messenger involved in regulation of a variety of cellular functions, including transcription. The Ca2+-bound form of the Ca2+-binding protein calmodulin (Ca2+/CaM) has been shown to inhibit DNA binding of E-proteins, but not tissue specific bHLH transcription factors, through direct physical interaction with the DNA binding basic sequence. The main focus of this thesis is on the role of Ca2+-binding proteins in regulation of bHLH transcription factors. Solution structure analysis of CaM in complex with the CaM-binding basic sequence of an E-protein revealed a novel type of protein-protein interaction with alternative binding modes in a complex of a CaM dimer surrounding the dimer of the E-protein sequence. This model for the interaction was further supported by mutational analysis, since every amino-acid substitution in the CaM binding basic sequence of E12 only partially affected the interaction with CaM. The mechanism of Ca2+/CaM regulation of transcriptional activation by E-proteins was studied using a cell culture system. CaM overexpression inhibited transcriptional activation by E12, E47 and SEF2-1 but not by MyoD. Ca2+/CaM inhibition of DNA binding in vitro directly correlated with the inhibitory effects of Ca2+ stimulation and CaM overexpression on transcription in vivo in a series of E12 basic sequence mutants. Furthermore, in vivo DNA binding of E12, but not a CaM resistant mutant of E12, was inhibited by overexpression of CaM. The data indicate that Ca2+/CaM can inhibit transcriptional activation by E-proteins through formation of a CaM-E-protein complex that can not bind DNA. An in vitro myogenesis system was used to investigate the potential role of the CaM-E-protein interaction in regulation of differentiation. CaM resistant mutants of E12 were inhibitory in MyoD initiated myogenic conversion of transfected fibroblasts, and inducers of intracellular Ca2+ activated, and Ca2+-channel blockers inhibited, transcriptional activation by E12, but not by a CaM resistant mutant of E12, with MyoD. The data support a model that Ca2+/CaM plays a role in initiation of myogenic differentiation through inhibition of E-protein dimers that can function as competitors to the CaM resistant MyoD/E-protein heterodimers required for myogenesis. The potential involvement of the Ca2+-binding calretinin proteins in regulation of bHLH transcription factors was also studied. Calretinin and the alternative splice variant calretinin-22k have been proposed to function as Ca2+-buffer proteins. Calretinin expression is restricted primarily to neuronal tissues. Calretinin and calretinin-22k are also found expressed in colon cancers, but not in normal colon tissue, and a role for calretinins in tumorigenesis has been proposed. We show that calretinins can inhibit DNA binding and transcriptional activation by E12 through basic sequence interaction. Endogenous E12/E47 and calretinin co-localize in a subset of cells in a proliferating colon cancer cell line and can be co-immunoprecipitated from the cell extract. A model is proposed in which calretinin overexpression can contribute to tumorigenesis through inhibition of the anti-proliferative function of E-proteins. The role of the E-protein E2-2 in lymphocyte development was studied using genetically altered mice with mosaic deletion of the E2-2 gene. The proportion of cells with a functional E2-2 allele was increased in the B- and T-lymphocyte populations, indicating a role for E2-2 not only in B-cell development, as reported before, but also in T-cell development.

Previous studies in Dr. Yang’s laboratory have established a grafting, design, and subdomain approach in order to investigate the properties behind Ca2+-binding sites located in Ca2+-binding proteins by employing engineered proteins. These approaches have not only enabled us to isolate Ca2+-binding sites and obtain their Ca2+-binding affinities, but also to investigate conformational changes and cooperativity effects upon Ca2+ binding. The focus of my thesis pertains to optimizing the expression and purification of engineered proteins with tailored functions. Proteins were expressed in E. coli using different cell strains, vectors, temperatures, and inducer concentrations. After rigorous expression optimization procedures, proteins were further purified using chromatographic and/or refolding techniques. Expression and purification optimization of proteins is essential for further analyses, since the techniques used for these studies require high protein concentrations and purity. Evaluated proteins had yields between 5-70 mg/L and purities of 80-90% as confirmed by SDS-PAGE electrophoresis.

Ca2+, a signal for death and life, is closely involved in the regulation of numerous important cellular events. Ca2+ carries out its function through its binding to Ca2+-receptors or Ca2+-binding proteins. The EF-hand protein, with a helix-loop-helix Ca2+-binding motif, constitutes one of the largest protein families. To facilitate our understanding of the role of Ca2+ in biological systems (denoted as calciomics) using genomic information, an improved pattern search method (http://www.chemistry.gsu.edu/faculty/Yang/Calciomics.htm) for the identification of EF-hand and EF-like Ca2+-binding proteins was developed. This fast and robust method allows us to analyze putative EF-hand proteins at the genome-wide level and further visualize the evolutionary scenario of the EF-hand protein family. This prediction method further enables us to locate a putative viral EF-hand Ca2+-binding motif within the rubella virus nonstructural protease that cleaves the nonstructural protein precursor into two active replicase components. A novel grafting approach has been used to probe the metal-binding properties of this motif by engineering the predicted 12-residue Ca2+-coordinating loop into a non-Ca2+-binding scaffold protein, CD2 domain 1. Structural and conformational studies were further performed on a purified, bacterially-expressed NS protease minimal metal-binding domain spanning the Zn2+- and EF-hand Ca2+-binding motif. It was revealed that Ca2+ binding induced local conformational changes and increased thermal stability. Furthermore, functional studies were carried out using RUB infectious cDNA clone and replicon constructs. Our studies have shown that the Ca2+ binding loop played a structural role in the NS protease and was specifically required for optimal stability under physiological conditions. In addition, we have predicted and characterized a calmodulin-binding domain in the gap junction proteins connexin43 and connexin44. Peptides encompassing the CaM binding motifs were synthesized and their ability to bind CaM was determined using various biophysical approaches. Transient expression in HeLa cells of two mutant Cx43-EYFP constructs without the putative CaM-binding site eliminated the Ca2+-dependent inhibition of gap junction permeability. These results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 and Cx44 in a Ca2+-dependent manner, providing a molecular basis for the well-characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Ca2+, both as a first and a second messenger, is closely involved in the modulation and regulation of numerous important cellular events, such as cell proliferation, differentiation and cell death. Fine-tuned Ca2+ signaling is achieved by its reversible or irreversible binding to a repertoire of Ca2+ signaling molecules. Among them, the extracellular calcium sensing receptor (CaSR) senses Ca2+ concentration ([Ca2+]o) in the milieu outside of cells where Ca2+ serves as a first messenger. An array of naturally-occurring mutations in CaSR has been found in patients with inherited disorders of Ca2+ homeostasis, leading to abnormal intracellular responses toward [Ca2+]o. In the present study, we have computationally predicted and experimentally characterized the metal-binding properties of five Ca2+-binding sites within CaSR and the accompanying metal--induced conformational changes by using two complementary methods-the grafting approach and the subdomain approach. Based on our results, a model has been proposed to explain the distinct CaSR-mediated responses toward abnormally ¡°high¡± or ¡°low¡± extracellular Ca2+ levels. In addition, we predicted and verified the interaction between CaSR with the most ubiquitously expressed four EF-hand-containing intracellular Ca2+ sensor protein, calmodulin (CaM). Our results demonstrate that the C-terminal CaM-binding domain of the CaSR is essential for proper intracellular Ca2+ response to external signals. Furthermore, we have applied the grafting approach to study the metal-binding properties and oligomeric state of the single EF-hand containing protein, STIM1. Our studies confirmed that the single EF-hand motif in STIM1, which resides in an equilibratium between its monomeric and dimeric forms, was capable of binding Ca2+ with a dissociation constant comparable to the ER Ca2+ concentration, suggesting it could function as a ER Ca2+ sensor responsible for sensing the Ca2+ filling state of ER.

Calmodulin (CaM) is a ubiquitous intracellular protein that regulates biological activities of numerous enzymes and ion channels. Upon responding Ca2+ concentration change, Ca2+- dependent CaM activates the hydrolyzation of cGMP by PDE and Ca2+ releasing channel activity of ryanodine receptor. In this dissertation, a series of CaM variants were engineered to enhance Ca2+ binding affinities by increasing the number of negative charged residues in individual EF-hand. The capability of shifting the biphasic Ca2+-activation profile of RyR1 is significantly altered by changing Ca2+ binding affinity of CaM at the C-terminal. This indicates that examining Ca2+-CaM affinity is a valid strategy to tune the activation profile of CaM-regulated ion channels. To further understand interactions between CaM and RyR1, NMR was used to determine their binding mode. To dissect roles of structural components of CaM in metal binding and regulation of biological functions of target proteins, we created half-CaMs and Del-CaM. Binding affinities of these variants to Ca2+, Tb3+ and Gd3+ were determined by fluorescence spectroscopy; functional studies were conducted using single channel analysis and PDE function assay. Another objective of my dissertation is to design a protein based contrast agent for molecular imaging. CaM was selected as the scaffold protein for designing Gd3+ based MRI contrast agent by modifying metal binding sites as well as grafting a biomarker peptide into the linker region to specifically target cancers with efficient and optimized modifications. The physical kinetic properties and animal imaging effects of these designed contrast agents were investigated by various methods.

Resultats anteriors mostraven que rates amb obesitat nutricional ("dieta de cafeteria") tenien un perfil metabòlic d'estalvi de nitrogen, mentre que pel model d'obesitat genètica (Zucker (fa/fa)) es suggeria un malbaratament de nitrogen. Es mostra una reducció en la taxa de recanvi d'alanina per l'animal sencer en el model d'obesitat nutricional, i un increment en el model d'obesitat genètica. La fracció anabòlica de la taxa és la responsable d'aquestes alteracions, suggerint perturbacions en el metabolisme de proteïnes. S'estudiaren músculs individuals, escollits per a assolir un rang ampli de perfils fibril•lars. L'obesitat genètica causa una reducció del contingut en proteïna, principalment en els músculs lents i oxidatius, la qual cosa correlaciona amb un increment en l'activitat µ-calpaïna, congruent amb una taxa de degradació de proteïnes incrementada Per contra, a l'obesitat nutricional, s'observa un increment en el contingut de proteïnes, fonamentalment en els músculs ràpids i glicolítics que no es va poder associar a variacions en l'activitat del sistema calpaïna, congruent amb una taxa de síntesi de proteïnes incrementada

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.

P68 RNA helicase (p68), as a DEAD family protein, is a typical RNA helicase protein. P68 functions in many other biological processes, which include the regulations of the gene transcription, cell proliferation and cell differentiation. In our group, Y593 phosphorylated p68 was found to have a function in the epithelial mesynchymal transition, which is an important process for cancer metastasis. In the present study, we found that p68 is a nucleocytoplasmic shuttling protein. The protein carries two functional nuclear exporting signal sequences and two nuclear localization signal sequences. Calmodulin, a calcium sensor protein, is well known to play roles in cell migration by regulating the activities of its target proteins at the leading edge. Calmodulin interacts with p68 at the IQ motif of p68. However, the biological function of this interaction is not known. In this study, we found that the p68/calmodulin protein complex functions as a microtubule motor in migrating cells. The shuttling function of p68 along with the motor function of p68/calmodulin causes the leading edge distribution of calmodulin in migrating cells. Disruption the interaction between p68 and calmodulin inhibits cancer cell metastasis in an established mouse model. On the other hand, Y593-Y595 double phosphorylated p68 were found to interact with PKM2, an important tumor isoform of pyruvate kinase. The shuttling function of p68 is reasoned to promote the dimer formation of PKM2 and transport the PKM2 to the cell nucleus. The nuclear PKM2 was found to function as a protein kinase to promote cell proliferation. In specific, the nuclear PKM2 phosphorylates and activates Stat3, an important transcription factor functions in cell proliferation. Overall, p68 is found to have functions in both cell migration and cell proliferation, and these two functions depend on the nucleocytoplasmic shuttling activity and the post-translational modification of p68.

Calmodulin (CaM) is a ubiquitous cytosolic Ca2+-binding protein involved in the binding and regulation of more than three-hundred intracellular target proteins. CaM consists of two globular domains joined by a central linker region. In the archetypical model of CaM binding to a target protein, the Ca2+-replete CaM wraps its two domains around a single α-helical target peptide; however, other conformations of CaM bound to target peptides and proteins have recently been discovered. Due to its ability to bind and affect many different intracellular processes, there is significant interest in a better understanding of the structural and conformational basis of CaM’s ability to bind and recognize target proteins. The mammalian nitric oxide synthase (NOS) enzymes are bound and activated by CaM. The NOS enzymes catalyze the production of nitric oxide (•NO), a free radical involved in numerous intercellular processes such as neurotransmission, vasodilation, and immune defense. There are three different isoforms of nitric oxide synthase (NOS) found in mammals – neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). All three enzymes are homodimeric with each monomer consisting of an N-terminal oxygenase domain and a multidomain C-terminal reductase domain. A CaM-binding domain separates the oxygenase and reductase domains. There is a unique opportunity to investigate CaM’s control over •NO production by the NOS enzymes since each isoform shows a different mode of activation and control by CaM. At elevated cellular Ca2+ concentrations, CaM is able to bind and activate nNOS and eNOS. In contrast, the iNOS isozyme is transcriptionally regulated and binds to CaM in the absence of Ca2+. The focus of this thesis is to better our present understanding of the conformational and structural basis for CaM’s ability to bind and activate the three mammalian NOS isozymes with particular emphasis on the interactions between CaM and iNOS. To further investigate the differences in the association of CaM to the Ca2+-dependent and Ca2+-independent NOS isoforms, a variety of CaM mutants including CaM-troponin C chimeras, CaM EF hand pair proteins, and CaM mutants incapable of binding to Ca2+ were employed. The inherent differences in binding and activation observed using these CaM mutants is described. Differences in the binding of the N- and C-terminal domains, as well as the central linker of CaM to peptides corresponding to the CaM-binding domain of each NOS enzyme and holo-NOS enzymes was investigated. The conformation of CaM when bound to NOS peptides and holo-NOS enzymes was also studied using fluorescence (Förster) resonance energy transfer (FRET). A preliminary three-dimensional structural study of Ca2+-replete and Ca2+-deplete CaM in complex with an iNOS CaM-binding domain peptide is also described. Combining the cumulative results in this thesis, a working model for iNOS’s regulation by CaM is proposed. Future suggested experiments are described to further the characterization of CaM binding to the NOS enzymes and other CaM-target proteins. The studies described in this thesis have expanded and improved the present understanding of the CaM-dependent binding and activation of the NOS isozymes, particularly the interactions between CaM and iNOS.

A major sweet potato leaf-type catalase was detected and identified from fullyexpandedmature leaves using in-gel activity staining assay with native- andSDS-PAGEs. The putative catalase activity band was inhibited by a catalaseinhibitor 3-amino-1,2,4-triazole. The major leaf-type catalase activity wasoptimal over 8, and was significantly repressed by £]-mercaptoethanol. However,its activity was much less affected by temperature within the range of 5 to 450C.Temporal and spatial expression showed that it was specifically detected inleaves, but not in roots and stems. Its activity increased from the immature L2leaves, and reached the maximal at the fully-expanded mature L3 leaves, thenslightly decreased in partial yellowing senescent L4 leaves, and was almost notdetected in completely yellowing L5 leaves similar to folding unopenedimmature L1 leaves. The catalase level showed approximately inversecorrelation with the H2O2 amounts in leaves of different developmental stages.Dark and ethephon, an ethylene-releasing compound, also temporarily enhancedthe catalase activities from 6 h to 24 h, however, the enhanced activitydecreased from 24 h to 48 h in detached leaves after treatment. The catalaselevel also showed approximately negative correlation with the H2O2 amounts intreated leaves. The major leaf-type catalase activity was repressed by EGTA,and the repression can be reversed by exogenous CaCl2. The major leaf-typecatalase activity was also repressed by calmodulin inhibitor chlorpromazine,and the repression can be reversed by exogenous purified SPCAM calmodulinfusion protein. Chlorpromazine-treated leaves also elevated H2O2 amount.Based on these data we conclude that a major leaf-type catalase with maximalactivity in L3 leaf was identified in sweet potato. Its activity was temporarilyenhanced by dark and ethephon, and was modulated by external calcium ion(Ca2+) and calmodulin. A possible physiological role and function in associationwith cellular H2O2 homeostasis in cope with developmental and environmentalcues in sweet potato leaves is suggested.